Figure 2. Composition and architecture of the actin fusion focus. (A) Homothallic h90 myo52-tdTomato GFP-CHD strain. Myo52 localizes as an intense dot at the cell–cell contact site, at the edge of the actin density. (B) Time-lapse imaging of homothallic h90 fus1-sfGFP pmap3:tdTomato strain. Entry of tdTomato in the h− cell is used as a marker for fusion. Fus1 is detected as an intense dot at the cell–cell contact site. (C) Homothallic h90 myo52-tdTomato fus1-sfGFP strain. Myo52 and Fus1 colocalize at the fusion site. (D) Homothallic h90 wild-type (left) and fus1Δ (right) strains expressing Myo52-GFP. Myo52 localizes as a crescent at the shmoo tip in the absence of Fus1. Cell outlines are shown with dotted lines. (E) Cross of heterothallic h+ myo52-tdTomato and h90 fus1Δ myo52-GFP. Myo52 forms a crescent in the fus1Δ cell and a dot in the wild-type cell. (F) 3D SIM time-lapse of GFP-CHD in homothallic h90 wild-type (WT), for3Δ, fus1Δ, and fus1Δ for3Δ mating pairs. Inverted images are shown. Green arrows point to actin filaments emanating from the fusion focus. No actin cables were detected in fus1Δ for3Δ double mutant, but some mating pairs showed a perinuclear actin ring (asterisks). (G) Mean length of actin filaments emanating from the fusion focus in strains as in F. Filaments are significantly shorter in for3Δ and longer in fus1Δ than wild-type cells (t test, ***, P < 10−6). This indicates that Fus1-dependent filaments are shorter than For3-dependent filaments and that wild-type cells likely contain both types. n = 30 actin filaments measured in three distinct mating pairs. Error bars are standard deviations. (H) Crosses of heterothallic h+ and h− myo52-tdTomato strains coexpressing Cdc12-3GFP, mEGFP-Cdc15, For3-3GFP, Myo51-3YFP, mGFP-Myo1, or Dip1-GFP. Images shown are time-averaged maximum intensity projection of 15 z stacks over 15 min. (I) Venn diagram summarizing the actin binding proteins that we show to be localized or not at the actin fusion focus. Attribution to cables, ring, or patches is adapted from Kovar et al. (2011). Bars, 1 µm.
Figure 2.

Composition and architecture of the actin fusion focus. (A) Homothallic h90 myo52-tdTomato GFP-CHD strain. Myo52 localizes as an intense dot at the cell–cell contact site, at the edge of the actin density. (B) Time-lapse imaging of homothallic h90 fus1-sfGFP pmap3:tdTomato strain. Entry of tdTomato in the h− cell is used as a marker for fusion. Fus1 is detected as an intense dot at the cell–cell contact site. (C) Homothallic h90 myo52-tdTomato fus1-sfGFP strain. Myo52 and Fus1 colocalize at the fusion site. (D) Homothallic h90 wild-type (left) and fus1Δ (right) strains expressing Myo52-GFP. Myo52 localizes as a crescent at the shmoo tip in the absence of Fus1. Cell outlines are shown with dotted lines. (E) Cross of heterothallic h+ myo52-tdTomato and h90 fus1Δ myo52-GFP. Myo52 forms a crescent in the fus1Δ cell and a dot in the wild-type cell. (F) 3D SIM time-lapse of GFP-CHD in homothallic h90 wild-type (WT), for3Δ, fus1Δ, and fus1Δ for3Δ mating pairs. Inverted images are shown. Green arrows point to actin filaments emanating from the fusion focus. No actin cables were detected in fus1Δ for3Δ double mutant, but some mating pairs showed a perinuclear actin ring (asterisks). (G) Mean length of actin filaments emanating from the fusion focus in strains as in F. Filaments are significantly shorter in for3Δ and longer in fus1Δ than wild-type cells (t test, ***, P < 10−6). This indicates that Fus1-dependent filaments are shorter than For3-dependent filaments and that wild-type cells likely contain both types. n = 30 actin filaments measured in three distinct mating pairs. Error bars are standard deviations. (H) Crosses of heterothallic h+ and h− myo52-tdTomato strains coexpressing Cdc12-3GFP, mEGFP-Cdc15, For3-3GFP, Myo51-3YFP, mGFP-Myo1, or Dip1-GFP. Images shown are time-averaged maximum intensity projection of 15 z stacks over 15 min. (I) Venn diagram summarizing the actin binding proteins that we show to be localized or not at the actin fusion focus. Attribution to cables, ring, or patches is adapted from Kovar et al. (2011). Bars, 1 µm.

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